Mild Alkaline Conditions Research Articles

Distinct Roles of Two Heme Centers for Transmembrane Electron Transfer in Cytochrome b561 from Bovine Adrenal Chromaffin Vesicles as Revealed by Pulse Radiolysis

The reaction of monodehydroascorbate (MDA) radical with purified cytochrome b561 from bovine adrenal chromaffin vesicles was investigated by the technique of pulse radiolysis. Radiolytically generated MDA radical oxidized rapidly the reduced form of cytochrome b561 to yield the oxidized form. Subsequently the oxidized form of cytochrome b561 was re-reduced by ascorbate in the medium. The second-order rate constants of the reaction of MDA radical were increased with decreasing pH, whereas a maximum of the second-order rate constant for the reaction with ascorbate was obtained around pH 6.8. At excess MDA radical to cytochrome b561 concentration, only half of the heme in cytochrome b561 was oxidized, indicating that only one of the two heme centers can react with MDA radical. On the other hand, when the reactions were examined using cytochrome b561 pretreated in a mild alkaline condition in the oxidized state, the cytochrome b561 could not be oxidized with MDA radical, suggesting that the heme center specific for the electron donation to MDA radical is selectively modified upon the alkaline treatment. These results suggest that the two heme b centers have distinct roles for the electron donation to MDA radical and the electron acceptance from ascorbate, respectively.

Photochemistry of 4-Thiothymine Derivatives in the Presence of N-9-Substituted-Adenine Derivatives: Formation of N-6-Formamidopyrimidines.

UV irradiation of aqueous solutions containing either 4-thiothymin-1-ylacetic acid (1b) and adenosine (2a), 4-thiothymidine (1a) and adenin-9-ylacetic acid (2b), or 1b and 2b led to 4,5-diamino-6-formamidopyrimidine (N-6-Fapy-Ade) derivatives as observed after irradiation of a mixture of 1a and 2a (J. Am. Chem. Soc. 1996, 118, 8142-8143). These new observations demonstrate that the replacement of one or both nucleoside sugar residues by a carboxymethyl group does not affect the regioselective course of the photochemical reaction. The thermal decomposition of 3a that resulted from irradiation of 1a in the presence of 2a, was examined along with its behavior under mild alkaline conditions. Finally, irradiation of N-3-methyl-4-thiothymidine (6a) in the presence of adenosine gave the N-3-methylcytidine derivative 7.

Bleach activation of thermomechanical pulp

AbstractUnder mild alkaline condition the bleaching activator tetraacetyl ethylenediamine (TAED) forms peroxyacetic acid with sodium perborate and improves the brightness of thermomechanical pulp. The activator makes it possible to bleach the pulp efficiently even without addition of sodium hydroxide at any temperature in the range 40 to 70°C. A mathematical model has been proposed to allow estimation of the total peroxy acid consumption during activated bleaching process. Using this model it is possible to calculate the extent of peroxy acid decomposition by predetermining the rate constant and activation energy. The effect of bleach activation was found to be maximum at temperatures below 60°C. The influences of bleaching time, pH, consistency, temperature and TAED charge on the bleach activation has been evaluated. The activator is effective enough to improve the brightness of sodium perborate bleached pulp at an average charge of about 50 mass% of sodium perborate. Apparently, high consistency dispersive bleaching without free alkali is a technological and economic advantage of the process.

Synthesis of 3α- and 3β-dimers from selected bile acids

New dimers of bile acids have been synthesized by linking the hydroxyl groups on C3 of the steroid skeleton with a spacer via the formation of ester or ether bonds. 3α-Dimers of lithocholic acid and cholic acid were prepared by forming ester linkages between the C3 hydroxyl group and dicarboxylic acids of different lengths, i.e., suberoyl chloride (eight carbons) and sebacoyl chloride (ten carbons). These dimers were obtained in three steps with an overall yield of about 70%. Regioselective hydrolysis of the methyl ester protecting groups on the bile acids was carried out successfully under mild alkaline conditions (aqueous lithium hydroxide with tetrahydrofuran). A 3β-dimer of lithocholic acid has also been synthesized by linking the C3 positions of two bile acids with diethylene glycol by the formation of ether linkages. The carboxylic acid groups of the bile acid dimer can be liberated by the removal of the methyl ester protecting groups.

The molecular weight distribution of polymerized iso-butylsilane and γ-methacryloxypropylsilane in aqueous solutions

The condensation of iso-butyltrimethoxysilane and γ-methacryloxypropyltrimethoxysilane (γ-MPS) in aqueous solutions has been studied. The silanes were hydrolysed in acidic solutions followed by adjustment of the pH of the solutions to allow condensation at different pH conditions. The molecular weights of the polymer mixtures obtained were determined by using gel permeation chromatography (GPC) with a light scattering detector. The GPC results suggest that the polybutylsilsesquioxanes differ in molecular weight distribution to the polymethacryloxypropylsilsesquioxanes. In general, polymers with the highest molecular weight were obtained in mild alkaline conditions. Strongly acidic as well as strongly alkaline conditions gave low molecular weight oligomers. The effect of fluoride ions on polymer molecular weight was also investigated. The polymers from the hydrolysis and condensation of iso-butylsilane and γ-MPS were monodisperse when the condensation was catalysed by fluoride ion. The molecular weights of the polymers were lower than those from the analogous base-catalysed reactions and the molecular weights increased with an increase in fluoride concentration.

Oligomeric flavanoids. Part 24. Controlled biomimetic synthesis of profisetinidin triflavanoid related phlobatannins

The complex structures of the economically important group of profisetinidin triflavanoid related phlobatannins are synthetically accessable in a controlled biomimetic fashion via the repetitive formation of the interflavanyl bond and pyran ring rearrangement of the chain extender unit under mild alkaline conditions. The repetitive formation of the interflavanyl bond and pyran ring rearrangement of the chain extender unit permit controlled synthetic access to phlobatannins at the ‘trimeric’ level [Display omitted]

Synthesis of [4,5-Bis(hydroxymethyl)-1,3-oxathiolan-2-yl]nucleosides as Potential Inhibitors of HIV via Stereospecific Base-Induced Rearrangement of a 2,3-Epoxy Thioacetate(1).

The synthesis of [4,5-bis(hydroxymethyl)-1,3-oxathiolan-2-yl]nucleosides is described. 2,3-Epoxy alcohol 10 was converted in one pot into thioacetate 11. Treatment of 11 under mild alkaline conditions gave thiirane 12 with inversion of configuration at C-2. We also found that thioacetate 11 rearranges into thiirane 14 under mild acidic conditions. This rearrangement reaction was shown by independent synthesis to proceed with net retention of configuration at C-2. We have proposed a tentative mechanism which may explain the results obtained. Opening of thiiranes 12 and 14 followed by deprotection gave (2R,3R)-2-thiothreitol (23) and (2S,3R)-2-thioerythritol (25), respectively. Regioselective silylation of the primary hydroxyl groups of 23 followed by treatment with trimethyl orthoformate gave 2-methoxy-1,3-oxathiolanes 26 and 27. Condensation with silylated bases followed by deprotection and separation of the anomers gave the oxathiolanylnucleosides. Compounds 29-31, 34, and 35 were found to be inactive when tested for inhibition of HIV-1 activity in vitro.

Structure revision and internal acyl migration of gymnemarsgenin

Reinvestigation of the NMR spectra of gymnemarsgenin by the selective INEPT and various two-dimensional NMR techniques (DQF-COSY, ROESY, HETCOR) led to the structural revision of gymnemarsgenin as 12 β- O-cinnamoyl-20- O-benzoyl sarcostin. The 1H and 13C NMR spectra were completely assigned. It is apparent that the 12-cinnamoyl group migrated to C-20 under the mild alkaline conditions rendered for partial saponification.

Analysis of the NADPH oxidase components during differentiation of HL-60 cells to eosinophilic lineage

HL-60 cells were induced to differentiate into eosinophil-like cells with sodium butyrate after passage under mild alkaline condition. The differentiating cells gradually possessed the Luxol-fast-blue (LFB) staining-positive granules and the capacity to produce superoxide. The increase in the amounts of cytochrome b-558 paralleled the superoxide anion generating activity. Immunoblot analysis demonstrated that p47-phox cytosolic oxidase protein appeared 1 day after differentiation, and increased up to 7 days. On the other hand, p67-phox cytosolic oxidase protein appeared in 3 days, and increased gradually up to 7 days. The oxidase activity did not appear until p67-phox protein was expressed in the cytosol during eosinophilic differentiation, indicating that p67-phox protein is likely to be a key protein of cytosolic factors also in eosinophilic differentiating cells. The amounts of p47-phox and p67-phox translocated to the plasma membrane in response to phorbol myristate acetate (PMA) increased with increasing amounts of cytochrome b-558 in the membrane. Our data demonstrate that the appearance of NADPH oxidase activity during eosinophilic differentiation is dependent on the levels of p47-phox and p67-phox cytosolic proteins translocated to the plasma membrane and the amount of cytochrome b-558 in the membrane as observed with neutrophils and monocytes.

In-Tube cDNA Cloning Method Using a Biotinylated RNA Probe

We report a simple, practical method for isolating a particular cDNA from a single-stranded (ss) cDNA library in a tube without using a radioisotope. The method consists of three steps: (i) the capture of a target cDNA by a biotinylated RNA via intermolecular hybrid formation, (ii) the binding of the cDNA-RNA hybrid to an avidin-coated gel, (iii) the recovery of the target cDNA by degradation of the RNA under mild alkaline conditions. The effectiveness of the method was examined by isolating a clone carrying a metapyrocatechase gene from a model library. This model experiment achieved an enrichment of 4800-fold. This method was applied to cloning of a cDNA encoding interleukin 8 (IL-8) from a cDNA library prepared from phorbol myristate acetate-treated U937 cells. Using an RNA probe prepared from a truncated cDNA encoding IL-8, the corresponding full-length cDNA clones were successfully obtained.

Synthesis of Peptides Influencing Growth Hormone Release

A solid phase peptide synthesis of 17 growth hormone (GH) releasing peptide analogues Ia - IIIc for their use in a pharmacological assay on GH release is described. While the linear peptide amides Ia - Ij were synthesized on p-methylbenzhydrylamine resin using Boc strategy, and cleaved by HF in the presence of scavengers the linear peptide amides Ik and Il were prepared on Merrifield benzyl ester type resin using Fmoc strategy and cleaved by ammonolysis. The deleted peptide amides IIa and IIb were obtained as by-products during HPLC purification of analogues Ic and Id. The linear precursors of cyclic peptides IIIa - IIIc were also prepared on Merrifield resin and cleaved under mild alkaline conditions. Their cyclization was performed in solution by diphenylphosphoryl azide.

The multiple hydroperoxides of choline phospholipids occurring in plasma after ischemia-reperfusion in rat liver.

Since phosphatidylcholine is a primary membrane component, and its peroxidation may result in disruption and dysfunction of the biomembrane, we used phosphatidylcholine hydroperoxide (PCOOH) as an index of oxidative-stress-related injury. We detected the occurrence of PCOOH in both liver and plasma from rats subjected to hepatic ischemia-reperfusion, and demonstrated that liver and plasma PCOOH levels reflect the extent of liver injury. However, the accuracy in measuring PCOOH levels, particularly in blood samples, is still in question since PCOOH may be rapidly eliminated by phospholipase A2 enzymes, which are widespread, easily activated, and have a preference for oxidized glycerophospholipids. When PCOOH levels were determined, using high-performance liquid chromatography to detect chemiluminescence (CL), the plasma samples showed a broad CL peak that began to elute at the same time as in the liver samples, but lasted longer. The peak appeared to contain hydroperoxides from multiple lipid classes. In addition to PCOOH, the hydroperoxides of sphingomyelin and lysophosphatidylcholine (SPHOOH and LPCOOH) were identified in the plasma of rats subjected to ischemia-reperfusion using thin layer chromatography separation and subsequent visualization, infrared spectroscopy, and hydrolysis under mild alkaline conditions. These results indicate that the occurrence of SPHOOH and LPCOOH, as well as PCOOH, may reflect liver damage related to ischemia-reperfusion.

Reactions of N-chloro β-lactams

N-Chloro β-lactam 4 was prepared to explore its potential for a methoxy transfer to the C3 position of the β-lactam. Methoxy addition to C3 of β-lactam 4 with concomitant loss of chloride to give α-methoxy β-lactam 5 occurred under mild alkaline conditions but was accompanied by methanolysis of the β-lactam ring as the major reaction pathway. Reaction of 4 with AgNO 3 promoted methoxy addition to C4 of the β-lactam followed by subsequent reactions.

Methylamine-treated low density lipoproteins elicit different responses in HepG2 cells and macrophages.

Recent results from this laboratory have demonstrated the existence of labile thiolester bonds in apolipoprotein B (ApoB). Thiolester bonds can be cleaved with nucleophiles such as methylamine, resulting in conformational change. The purpose of this study was to explore whether the cellular interactions would be altered after methylamine treatment of low density lipoproteins (LDL). Human hepatoma cells, HepG2, and human monocyte derived macrophages were used for these studies. Fresh LDL were incubated with methylamine under mild alkaline conditions under N2 and with preservatives for 24 h. The methylamine-treated LDL showed particle size and net charge identical to fresh native LDL. In addition, no oxidative modification of LDL occurred under the experimental conditions. The methylamine-treated LDL were indistinguishable from native LDL in HepG2 cells as judged by binding, degradation, cholesterol accumulation and de novo sterol synthesis. However, methylamine-treated LDL caused an increased accumulation of cholesteryl esters in macrophages which was comparable to the accumulation caused by acetylated LDL. Dual color digital imaging fluorescence microscopy revealed no competition between acetylated and methylamine-treated LDL, suggesting that the excessive uptake of methylamine-treated LDL was not mediated by the 'scavenger' receptor. The increased accumulation of cholesteryl ester in macrophages also did not appear to stem from the classical LDL receptor. These results suggest that a new receptor binding domain is exposed due to the conformational change upon treatment of LDL with methylamine.

A new type of safety-catch linker cleaved by intramolecular activation of an amide anchorage and allowing aqueous processing in solid-phase peptide synthesis

The N3-phenyloxycarbonyl-L-2,3-diaminopropionic acid residue behaves as a versatile iinker [Dpr(Phoc)linker] displaying high stability under neutral and acidic conditions but undergoes activation under mild alkaline conditions for the release of peptide acids or amides by nucleophilic cleavage.

One-hour downward alkaline capillary transfer for blotting of DNA and RNA

The downward alkaline capillary transfer of DNA and RNA from agarose gel to a hybridization membrane was performed using a transfer solution containing 3 m NaCl and 8 m m NaOH. Under mild alkaline conditions, DNA and RNA were completely eluted from the agarose gel and bound to a hybridization membrane within 1 h. On the basis of this new method of transfer a blotting protocol, downward alkaline blotting, was elaborated. It provides a fast and efficient alternative to commonly used Southern and Northern blotting protocols. The downward alkaline blotting of DNA and RNA can be completed in 2.5 and 1.5 h, respectively, and can be used with both plastic and nitrocellulose membranes. In addition, the downward alkaline blotting protocol allows for a hybridization efficiency of DNA and RNA higher than that of the standard blotting protocols performed at neutral pH.

A novel approach for chemically deglycosylating O-linked glycoproteins. The deglycosylation of submaxillary and respiratory mucins.

A new approach for removing O-glycosidically linked carbohydrate side chains from glycoproteins is described. Periodate oxidation of the C3 and C4 carbons in peptide-linked N-acetylgalactosamine (GalNAc) residues generates a dialdehyde product which, under mild alkaline conditions, undergoes a beta-elimination which releases carbohydrate and leaves an intact peptide core. The pH and time dependence, and intermediates of the elimination, have been extensively followed by carbon-13 NMR spectroscopy and amino acid analysis using ovine submaxillary mucin (OSM) as the substrate. The deglycosylation of OSM is complete and provides apomucin in high yield with an amino acid composition identical to the starting material. Carboxymethylated OSM when deglycosylated by this method gives an apomucin with an apparent molecular weight of ca. 700 x 10(3). The molecular weight is the same as that calculated for the peptide core of the starting mucin, demonstrating the absence of peptide core cleavage. This contrasts with the use of trifluoromethanesulfonic acid (TFMSA), which generates apomucin products of lower molecular weights. Oligosaccharide side chains substituted at C3 of the peptide-linked GalNAc residue are resistant to the oxidation and elimination. Glycoproteins containing these more complex side chains can be deglycosylated by pretreatment with TFMSA under mild (0 degree C) conditions, which removes peripheral sugars (while leaving the peptide-linked GalNAc residue intact), followed by oxidation and beta-elimination. Studies on the deglycosylation of porcine submaxillary mucin and human tracheobronchial mucin indicate that this approach provides more efficient removal of carbohydrate and less peptide core degradation than a more vigorous (25 degrees C) treatment with TFMSA alone. 13C NMR spectroscopic studies and carbohydrate analysis of the deglycosylation intermediates of the human mucin indicate that certain sialic acid containing and N-acetylglucosamine-containing oligosaccharides have elevated resistance to TFMSA treatment at 0 degrees C. By the use of neuraminidase, repeated mild TFMSA treatments, and multiple oxidations and beta-eliminations, the human mucin can be nearly completely deglycosylated. It is expected that all mucins and most glycoproteins containing O-glycosidic linkages can be readily and nearly completely deglycosylated using this combined approach.

Analysis of disulphide bridge function in recombinant bovine prolactin using site-specific mutagenesis and renaturation under mild alkaline conditions: a crucial role for the central disulphide bridge in the mitogenic activity of the hormone.

We have previously described a method for isolating Escherichia coli-produced methionyl bovine prolactin (Met-bPRL) and its renaturation using thioredoxin. This report describes an alternative renaturation procedure in which extracted Met-bPRL is incubated in air at pH 10 and 20 degrees C. Within 1 h of such treatment essentially all of the reduced Met-bPRL was converted to the oxidized form; this was accompanied by an increase to full mitogenic activity in the Nb2 cell bioassay. It was also found that, to minimize contamination by high mol. wt Met-bPRL derivatives, it is essential to have a reducing agent (dithiothreitol) present during disruption of the bacteria and to extract the protein at neutral pH. The contribution of each of the three disulphide bridges in bPRL to its bioactivity was studied with Met-bPRL variants, prepared via site-specific mutagenesis, in which cysteines were replaced by serines to prevent disulphide bond formation. Variants lacking the C4-C11 bridge, the C191-C199 bridge or both these terminal bridges were as mitogenic as authentic bPRL. (Variants lacking the C191-C199 bridge had markedly increased solubility in the presence of deoxycholate.) In contrast, variants lacking the C58-C174 bridge had greatly reduced bioactivity, indicating that integrity of the large disulphide loop is crucial to the hormone's mitogenic activity.

Studies on metal complexes of chiral cyclen. Part 14. Configurational isomerism in a complex of cobalt(III)

The chiral 12-membered cyclic tetramine (2R,5R,8R,11R)-2,5,8,11-tetraethyl-1,4,7,10-tetraazacyclododecane (L) reacts with CoBr2 to give mainly cis-SSSR-[CoIIIBr(H2O)L]Br21. Another isomeric complex 2 has recently been isolated from the reaction mixture obtained under mild alkaline conditions. In order to clarify the geometry, the structures of cis-(RSRS)-[Co{(S)-alaO}L][ClO4]24(alaO = alaninate) and cis-(RSRS)-[Co{(S)-thrO}L][ClO4]2·3H2O 6(thrO = threoninate) derived from 2 have been determined by X-ray analysis. All complexes prepared are cis-octahedral, each CoIII is surrounded by four N from L and by N and O of the amino acid, and the configurations of the four asymmetric nitrogen atoms are RSRS. This means that the cobalt ion in 2 co-ordinates to the crowded face of the N4 plane of L, and the ligations of the metal to L in 1 and 2 occur from opposite directions. The absorption band maxima of the respective visible spectra of all the RSRS complexes shift approximately 400 cm–1 to higher energy than those of the corresponding SSSR ones. A slow exchange reaction between the fifth and sixth ligands is observed for 1, and release of the amino acid from a SSSR-amino acidato complex takes place in Na2CO3 solution. No such reactions occur in 2 and in RSRS-amino acidato complexes. These results are probably related to the ligand-field strength.

Structural analysis of the N-linked oligosaccharides from murine glycophorin

Glycophorins, isolated from BALB/c mouse erythrocytes, were degraded under mild and strong reductive alkaline conditions and the N-linked Oligosaccharides were isolated as alditols. The oligosaccharide alditols were fractionated and purified using gel filtration, concanavalin A-Sepharose affinity chromatography, and high-performance ion-exchange chromatography. Structural analysis was carried out by chemical analyses, periodate oxidation in combination with fast atom bombardment mass spectrometry, and 500-MHz 1H NMR spectroscopy. The results revealed the presence of sialylated biantennary, triantennary, and tetraantennary complex type oligosaccharides, all fucosylated at the innermost N-acetylglucosamine residue. The tri- and tetraantennary oligosaccharide-containing fractions also contained species elongated by one and/or two N-acetyllactosamine (-3Galβ1-4GlcNAcβ1-) sequences. The N-linked oligosaccharides were shown to be combined only with one (the low molecular weight) of the two mouse glycophorins.